Substrate specificity of human collagenase 3 assessed using a phage-displayed peptide library

The substrate specificity of human collagenase 3 (MMP-13), a member of the matrix metalloproteinase family, is investigated using a phage-displayed ra ndom hexapeptide Library containing 2 x 10(8) independent recombinants, A t otal of 35 phage clones that express a peptide sequence that can be hydroly zed by the recombinant catalytic domain of human collagenase 3 are identifi ed. The translated DNA sequence of these clones reveals highly conserved pu tative P1, P2, P3 and P1', P2', and P3' subsites of the peptide substrates, Kinetic analysis of synthetic peptide substrates made from human collagena se 3 selected phage clones reveals that some of the substrates are highly a ctive and selective. The most active substrate, 2,4-dinitrophenyl-GPLGMRGL- NH2 (CP), has a k(cat)/K-m value of 4.22 x 10(6) M-1 s(-1) for hydrolysis b y collagenase 3, CP was synthesized as a consensus sequence deduced from th e preferred sub sites of the aligned 35 phage clones. Peptide substrate CP is 1300-, 11-, and 820-fold selective for human collagenase 3 over the MMPs stromelysin-l, gelatinase B, and collagenase 1, respectively. In addition, cleavage of CP is 37-fold faster than peptide NF derived from the major MM P-processing site in aggrecan, Phage display screening also selected five s ubstrate sequences that share sequence homology with a major MMP cleavage s equence in aggrecan and seven substrate sequences that share sequence homol ogy with the primary collagenase cleavage site of human type II collagen. I n addition, putative cleavage sites similar to the consensus sequence are f ound in human type N collagen. These findings support previous observations that human collagenase 3 can degrade aggrecan, type Il and type IV collage ns.